Quant-iT dsdna High-Sensitivity Assay Kit

Transcription

1 Quant-iT dsdna High-Sensitivity Assay Kit Table 1. Contents and storage information. Material Amount Concentration Storage Stability Quant-iT dsdna HS reagent (Component A) λ dsdna HS standards (Component C) Quant-iT dsdna HS buffer (Component B) 1.0 ml 200X in DMSO set of 8, 500 µl each 250 ml NA 0, 0.5, 1, 2, 4, 6, 8, and 10 ng/µl 6 C * Protect from light When stored as directed, kit contents are stable for at least 6 months. 6 C Protect from light * For convenience, the Quant-iT dsdna HS reagent may be stored indefinitely at room temperature, protected from light. For short-term storage (days), the buffer may be left at room temperature; however, for longer periods we recommend storage at 6 C to prevent microbial contamination. Number of labelings: 1,000, with a 200 µl assay volume in a 96-well microplate format. The Quant-iT dsdna HS assay can be adapted for use in cuvettes or 384-well microplates. Approximate fluorescence excitation/emission maxima: 502/523 nm Introduction The Quant-iT dsdna High-Sensitivity Assay Kit makes DNA quantitation easy and accurate. The kit provides concentrated assay reagent, dilution buffer, and pre-diluted DNA standards. Simply dilute the reagent 1:200, load 200 μl into the wells of a microplate, add 1 20 μl sample volumes, mix, then read the fluorescence. The assay is highly selective for double-stranded DNA over RNA, and in the range of ng, the fluorescence signal is linear with DNA (Figure 1). The assay is performed at room temperature, and the signal is stable for 3 hours. Common contaminants, such as salts, solvents, detergents, or protein are well tolerated in the assay. In addition to the Quant-iT dsdna High-Sensitivity Assay Kit described here, Molecular Probes offers the Quant-iT dsdna Broad-Range Assay Kit (Q33130). The Quant-iT dsdna Broad-Range Kit is designed for assaying samples containing ng of DNA. If you would like to use this kit with the Qubit fluorometer, we have included instructions under Using the Quant-iT dsdna High-Sensitivity Assay Kit with the Qubit Fluorometer. Revised: 28 February 2007 MP 33120

2 Figure 1. DNA selectivity and sensitivity of the Quant-iT dsdna HS assay. Triplicate 10 μl samples of l DNA ( ), E. coli rrna ( ), or a 1:1 mixture of DNA and RNA ( ) were assayed in the Quant-iT dsdna HS assay. Fluorescence was measured at 485/530 nm and plotted versus the mass of nucleic acid for the DNA alone or RNA alone, or versus the mass of the DNA component in the 1:1 mixture. The variation (CV) of replicate DNA determinations was 2%. The inset, a separate experiment with octuplicate determinations, shows the extreme sensitivity of the assay for DNA. Background fluorescence has not been subtracted. Experimental Protocol Handling the Quant-iT Reagent We must caution that no data are available addressing the mutagenicity or toxicity of the Quant-iT dsdna HS reagent. This reagent is known to bind nucleic acid and is provided as a solution in DMSO; treat the reagent with the same safety precautions as all other potential mutagens and dispose of the dye in accordance with local regulations. Remove the Quant-iT dsdna High-Sensitivity Assay Kit from storage and allow the components to equilibrate to room temperature. During all steps, protect the Quant-iT dsdna HS reagent concentrate and the working solution from light as much as possible. Figure 2. The Quant-iT dsdna High-Sensitivity assay. Quant-iT dsdna High-Sensitivity Assay Kit 2

3 Using the Quant-iT dsdna High-Sensitivity Assay Kit with a Fluorescence Microplate Reader This protocol describes the use of the Quant-iT dsdna High-Sensitivity Assay Kit with a fluorescence microplate reader equipped with excitation and emission filters appropriate for fluorescein or Alexa Fluor 488 dye. Some contaminating substances may interfere with the assay. See the Appendix for more information. For an overview of this procedure, see Figure Make a working solution by diluting Quant-iT dsdna HS reagent 1:200 in Quant-iT dsdna HS buffer. For example, for ~100 assays put 100 μl of Quant-iT dsdna HS reagent (Component A) and 20 ml of Quant-iT dsdna HS buffer (Component B) in a disposable plastic container and mix well. Do not use glass containers. Do not use buffers other than the Quant-iT dsdna HS buffer to make the working solution. 1.2 Load 200 μl of the working solution into each microplate well. Diluted Quant-iT dsdna HS reagent is stable for at least 3 hours at room temperature, protected from light. 1.3 Add 10 μl of each λ DNA standard (Component C) to separate wells and mix well. Take care not to introduce nucleases into the tubes of DNA standard as you remove aliquots for the assay. Duplicates or triplicates of the standards are recommended. 1.4 Add 1 20 μl of each unknown DNA sample to separate wells and mix well. Duplicates or triplicates of the unknown samples are recommended. Some contaminating substances may interfere with the assay, see the Appendix. 1.5 Measure the fluorescence using a microplate reader (excitation/emission maxima are 510/527 nm, see Figure 3). Standard fluorescein wavelengths (excitation/emission at ~480/530 nm) are appropriate for this dye. The fluorescence signal is stable for 3 hours at room temperature. 1.6 Use a standard curve to determine the DNA amounts. For the λ DNA standards, plot amount vs. fluorescence, and fit a straight line to the data points. Figure 3. Excitation and emission maxima for the Quant-iT dsdna HS reagent bound to DNA. Quant-iT dsdna High-Sensitivity Assay Kit 3

4 Data Analysis Considerations Standard Curves and Extended Ranges The fluorescence of the Quant-iT dsdna HS reagent bound to dsdna is extremely linear from ng. For best results at the low end of the standard curve, the line should be forced through the background point (or through zero, if background has been subtracted). When 10 μl volumes of the standards are used, the lowest DNA-containing standard represents 5 ng of DNA; nevertheless, highly accurate determinations of DNA down to 0.2 ng are attained using the standard curve as described above. To assess the reliability of the assay in the low range, use smaller volumes of the standards, e.g. 2 μl volumes for a standard curve ranging from 0 20 ng (Figure 4A). Alternatively, dilute the standards in buffer for an even-tighter range (Figure 4A, inset). During development of the Quant-iT dsdna HS assay, Molecular Probes scientists were able to detect 0.05 ng of λ DNA under ideal experimental circumstances (using calibrated pipettors, octuplicate determinations, the best microplate readers, and Z-factor 1 analysis). Your results may vary. If desired, the utility of the Quant-iT dsdna HS assay can be extended beyond 100 ng, up to 200 ng (Figure 4B). For standards in this range, use 20 μl volumes of the provided standards. Note that the standard curve may not be linear in the range ng, and high levels of RNA may now interfere slightly with the results. Figure 4. Extended ranges for the Quant-iT dsdna HS assay. Triplicate 2 μl (Panel A) or 20 µl samples (Panel B) of l DNA ( ), E. coli rrna ( ), or a 1:1 mixture of DNA and RNA ( ) were assayed in the Quant-iT dsdna HS assay. Fluorescence was measured at 485/530 nm and plotted versus the mass of nucleic acid for the DNA alone or RNA alone, or versus the mass of the DNA component in the 1:1 mixture. The inset (Panel A), a separate experiment with octuplicate determinations, shows the sensitivity of the assay for DNA. Background fluorescence has not been subtracted. Experimental Protocol Handling the Quant-iT Reagent We must caution that no data are available addressing the mutagenicity or toxicity of the Quant-iT dsdna HS reagent. This reagent is known to bind nucleic acid and is provided as a solution in DMSO; treat the reagent with the same safety precautions as all other potential mutagens and dispose of the dye in accordance with local regulations. Remove the Quant-iT dsdna High-Sensitivity Assay Kit from storage and allow the components to equilibrate to room temperature. During all steps, protect the Quant-iT dsdna HS reagent concentrate and the working solution from light as much as possible. Quant-iT dsdna High-Sensitivity Assay Kit

5 Using the Quant-iT dsdna High-Sensitivity Assay Kit with the Qubit Fluorometer The Quant-iT dsdna HS Assay Kit can easily be adapted for use with the Qubit fluorometer. The protocol below is abbreviated from the Qubit fluorometer instruction manual, which is available at probes.invitrogen.com/qubit. Although a step-by-step protocol and critical assay parameters are given here, more detail is available in the Qubit fluorometer instruction manual and you are encouraged to familiarize yourself with this manual before you begin your assay. See Figure 5 for an overview of the procedure. Figure 5. Overview for using the Quant-iT dsdna HS assay in the Qubit fluorometer. IMPORTANT: Ensure all assay reagents are at room temperature before you begin. 2.1 Label the lids of the assay tubes* you will need for the standards and user samples. Note: The Quant-iT dsdna HS Assay Kit requires two standards for calibration. You will prepare a dilution of the 0 ng/µl λ dsdna HS standard from the Component C set to generate Standard #1. You will prepare a dilution of the 10 ng/µl λ dsdna HS standard from the Component C set to generate Standard #2 (see step 2.3 below). 2.2 Make the Quant-iT dsdna HS working solution by diluting the Quant-iT dsdna HS reagent 1:200 in Quant-iT buffer. 2.3 Prepare assay tubes according to Table 2 below. Table 2. Tube setup. Standard assay tubes Volume of working solution (from step 2.2) to add 190 μl μl Volume of Standard (from kit) to add; [prepare Standard #1 by diluting 10 µl of the 0 ng/µl standard and Standard #2 by diluting 10 µl of the 10 ng/µl standard] 10 μl Volume of User Sample to add 1 20 μl Total volume in each assay tube 200 μl 200 μl User Sample assay tubes Quant-iT dsdna High-Sensitivity Assay Kit

6 2.4 Vortex all tubes for 2 3 seconds. 2.5 Incubate the tubes for 2 minutes at room temperature. 2.6 Calibrate the Qubit fluorometer using Standard #1 and Standard # Read the user samples in the Qubit fluorometer. 2.8 Multiply by the value given by the dilution factor (see the Qubit fluorometer instruction manual) to determine concentration of your original sample. Alternatively, choose Calculate sample concentration to have the Qubit fluorometer perfomr this multiplication for you (available on Qubit fluorometers with firmware version 1.22 or later). * Use only thin-wall, clear 0.5 ml PCR tubes. Acceptable tubes include Qubit assay tubes (500, Invitrogen Cat. no. Q32856) or Axygen PCR- 05-C tubes (VWR, part number ). Critical Assay Parameters for the Qubit Fluorometer Assay Temperature The Quant-iT dsdna HS assay for the Qubit fluorometer delivers optimal performance when all solutions are at room temperature. The Quant-iT assays were designed to be performed at room temperature, as temperature fluctuations can influence the accuracy of the assay (see Figure 2 in the Appendix of the Qubit fluorometer instruction manual). To minimize temperature fluctuations, store the Quant-iT dsdna HS reagent and the Quant-iT dsdna HS buffer at room temperature and insert all assay tubes into the Qubit fluorometer only for as much time as it takes for the instrument to measure the fluorescence, as the Qubit fluorometer can raise the temperature of the assay solution significantly, even over a period of a few minutes. Do not hold the assay tubes in your hand before reading, as this will warm the solution and result in a low reading. Incubation Time In order to allow the Quant-iT dsdna HS assay to reach maximum fluorescence, incubate the assay tubes for 2 minutes after mixing the sample or standard with the working solution. After this incubation period, the fluorescence signal is stable for 3 hours at room temperature. Photobleaching of the Quant-iT Reagent The Quant-iT dsdna HS reagent exhibits high photostability in the Qubit fluorometer, showing <0.3% drop in fluorescence after 9 readings and <2.5% drop in fluorescence after 40 readings. It is important to remember, however, that if the assay tube remains in the Qubit fluorometer for multiple readings, a temporary reduction in fluorescence will be observed as the solution increases in temperature (see Figure 2 in the Appendix of the Qubit fluorometer instruction manual). (The temperature inside the Qubit fluorometer may be as much as 3ºC above room temperature after 1 hour.) For this reason, if you want to perform multiple readings of a single tube, you should remove the tube from the instrument and let it equilibrate to room temperature for 30 seconds before taking another reading. Assay Tubes for Use with the Qubit Fluorometer Only thin-wall, clear 0.5 ml PCR tubes are appropriate for use in the Qubit fluorometer. Acceptable tubes include Qubit assay tubes (Invitrogen Cat. no. Q32856, 500 tubes) or Axygen PCR-05-C tubes (VWR, part number ). The assay volume must be 200 µl for an accurate read. Quant-iT dsdna High-Sensitivity Assay Kit

7 Calibrating the Qubit Fluorometer When quantitating your samples using the Qubit fluorometer, you have the choice to calibrate the instrument using freshly prepared calibration solutions or to apply the values from a previously run calibration. Using the Quant-iT dsdna High-Sensitivity Assay Kit with the Qubit Fluorometer above describes the preparation of fresh calibration standards. Consult the instruction manual for the Qubit fluorometer for guidance on choosing a calibration mode. Calculating the Concentration of Your Sample The Qubit fluorometer gives values in either μg/ml or ng/ml. This value corresponds to the concentration after your sample was diluted into the assay tube. To calculate the concentration of your sample, use the equation in the Qubit fluorometer instruction manual. Contaminating Substances Some contaminating substances may interfere with the assay. See the Appendix for more information. Appendix Contaminating Substances A number of common contaminants have been tested in the Quant-iT dsdna HS assay, and most are well tolerated (Table 3). For untested contaminating substances, and, in general, for highest accuracy, the standards should be assayed under the same conditions as the unknowns. For example, if the experimental samples are in an unusual buffer and if 10 µl volumes of these samples are used, then add 10 µl volumes of the unusual buffer (lacking DNA) to the assays of the standards. Table 3. Effect of Contaminants in the Quant-iT dsdna High-Sensitivity Assay. * Contaminant Final Concentration in the Assay Concentration in 20 μl Sample Concentration in 10 μl Sample Sodium chloride 50 mm 500 mm 1 M OK Result Magnesium chloride 5 mm 50 mm 100 mm OK Sodium acetate 30 mm 300 mm 600 mm OK Ammonium acetate 50 mm 500 mm 1 M OK Ethanol 1% 10% 20% OK Phenol 0.1% 1% 2% OK Chloroform 1% 10% 20% OK SDS 0.01% 0.1% 0.2% OK Triton X % 0.1% 0.2% OK dntps 100 μm 1 mm 2 mm OK BSA 10 mg/ml 100 mg/ml 200 mg/ml OK IgG 0.5 mg/ml 5 mg/ml 10 mg/ml OK * DNA standards were assayed in the presence or absence of contaminants at the indicated final concentrations. Equivalent concentrations (approximate) in 20 μl or 10 μl sample volumes are also listed. In all cases, results are given as OK, usually less than 10% perturbation. An acceptable result, but with some distortion of the standard curve; for best results, add the same amount of contaminant to the standard samples. Immiscible. A mixture of datp, dctp, dgtp, and dttp. Quant-iT dsdna High-Sensitivity Assay Kit 7

8 Reference 1. J Biomol Screen 4, (1999). Product List Current prices may be obtained from our website or from our Customer Service Department. Cat # Product Name Unit Size Q33120 Quant-iT dsdna Assay Kit, High Sensitivity, 1000 assays * ng* kit Contact Information Molecular Probes, Inc Willow Creek Road Eugene, OR Phone: (541) Fax: (541) Customer Service: 6:00 am to 4:30 pm (Pacific Time) Phone: (541) Fax: (541) probesorder@invitrogen.com Toll-Free Ordering for USA: Order Phone: (800) Order Fax: (800) Technical Service: 8:00 am to 4:00 pm (Pacific Time) Phone: (541) Toll-Free (800) Fax: (541) probestech@invitrogen.com Invitrogen European Headquarters Invitrogen, Ltd. 3 Fountain Drive Inchinnan Business Park Paisley PA4 9RF, UK Phone: +44 (0) Fax: +44 (0) euroinfo@invitrogen.com Technical Services: eurotech@invitrogen.com Further information on Molecular Probes products, including product bibliographies, is available from your local distributor or directly from Molecular Probes. Customers in Europe, Africa and the Middle East should contact our office in Paisley, United Kingdom. All others should contact our Technical Service Department in Eugene, Oregon. Molecular Probes products are high-quality reagents and materials intended for research purposes only. These products must be used by, or directly under the supervision of, a technically qualified individual experienced in handling potentially hazardous chemicals. Please read the Material Safety Data Sheet provided for each product; other regulatory considerations may apply. Limited Use Label License No. 223: Labeling and Detection Technology The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. The buyer may transfer information or materials made through the use of this product to a scientific collaborator, provided that such transfer is not for any Commercial Purpose, and that such collaborator agrees in writing (a) to not transfer such materials to any third party, and (b) to use such transferred materials and/or information solely for research and not for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture, use or sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed, provided that neither this product nor any of its components was used in the manufacture of such product. If the purchaser is not willing to accept the limitations of this limited use statement, Invitrogen is willing to accept return of the product with a full refund. For information on purchasing a license to this product for purposes other than research, contact Molecular Probes, Inc., Business Development, Willow Creek Road, Eugene, OR 97402, Tel: (541) Fax: (541) Several Molecular Probes products and product applications are covered by U.S. and foreign patents and patents pending. All names containing the designation are registered with the U.S. Patent and Trademark Office. Copyright 2007, Molecular Probes, Inc. All rights reserved. This information is subject to change without notice. Quant-iT dsdna High-Sensitivity Assay Kit 8